Abstracts

Rationale: Lacosamide (LCM, SPM 927, Vimpat) is an antiepileptic drug recently approved in the USA and Europe as add-on treatment for partial onset seizures. LCM presents a novel mode of action by selectively enhancing sodium channel slow inactivation without affecting fast inactivation which differentiates it from classical antiepileptic drugs (Errington et al., 2008). Initial investigations proposed that LCM might have an additional mode of action by interacting with the collapsin response mediator protein CRMP-2 (Beyreuther et al., 2007). In the present study we describe the investigation of LCM binding to the cloned human CRMP-2 protein using different biochemical and biophysical experimental approaches. Methods: The human CRMP-2 protein was transiently expressed with or without tags (6xHis, GST) in a variety of mammalian cells (HEK, COS) and Xenopus laevis oocytes (mRNA injection). Membrane fractions were prepared and CRMP-2 protein levels were verified by Western Blot. Experiments carried out on isolated CRMP-2 protein used tagged hCRMP-2 (His-C_t; GST-N_t) that was expressed and affinity purified from E. coli and coupled to scintillation proximity assay (SPA) beads. Specific binding of [³H]LCM (specific activity: 31 Ci/mmol, free concentration range: 100-1450 nM) was assessed on isolated and membrane bound CRMP-2 protein using classical filtration binding assays or scintillation proximity assays. Non-specific binding was determined in the presence of 1 mM LCM. The binding of unlabelled LCM to the histidine-tagged hCRMP-2 was investigated by surface plasmon resonance (Biacore, GE Healthcare) using a CM5 sensor chip. Results: The hCRMP-2 protein was efficiently expressed in membrane fractions from mammalian cells and Xenopus oocytes (~500ng/100 g membranes) and binding of [³H]LCM was investigated under various experimental conditions (25 C or 37 C; different buffer compositions). We observed no specific binding of [³H]LCM (free concentration 100-1450nM) to the hCRMP-2 protein expressed in membrane fractions or to the isolated tagged hCRMP-2 protein coupled to SPA beads under all experimental conditions tested. Biacore analysis showed that LCM over a concentration range of 0.39-100 M does not specifically bind to the hCRMP-2 protein. Conclusions: Previous investigations, using [¹⁴C]LCM, indicated that LCM may bind to the hCRMP-2 protein with a dissociation constant of ~5 M (Beyreuther et al., 2007). In this study we attempted to further characterize this putative binding site of LCM by using either [³H]LCM (having 1000 fold higher specific activity than [¹⁴C]LCM) or by measuring direct binding of unlabelled LCM to purified hCRMP-2 using surface plasmon resonance. Although these methods were well suited to measure binding affinities in the molar range, the results obtained were all negative. In conclusion, the present study, using a wide variety of experimental approaches, does not support the presence of a specific binding site for LCM on the hCRMP-2 protein. References: Beyreuther et al. CNS Drug Reviews 2007;13(1):21 & Errington et al. Mol Pharmacol. 2008;73:157